Recovery of Tissue Function Following Administration of B Cells to Injured Tissue

ABSTRACT

The present invention relates generally to systems and methods of enhancing recovery of function of injured tissue through administration of a composition comprising a relatively pure populations of B lymphocyte cells in a pharmaceutically acceptable carrier to the injured tissue. Kits are provided to aid in purification of B cells from heterogeneous mixtures of cells and administration of B cells to injured tissue.

PRIOR RELATED APPLICATIONS

The present applications claims the benefit of priority to U.S.provisional patent application Ser. No. 60/672,416 filed Apr. 18, 2005,which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to methods of enhancing recoveryof tissue function following injury through administration of relativelypure populations of B lymphocytes to injured tissue.

BACKGROUND

Loss of tissue function, whether by disease or accident, remains a majorhealth problem. Heart and brain injuries, for example, are two of theleading causes of death and disability throughout the world. In theUnited States, cardiac disease accounts for 40% of all deaths and is theleading cause of congestive heart failure (American Heart Association.Heart and Stroke Update. Dallas, Tex.: American Heart Association;2003). Cardiac disease that leads to acute myocardial infarction orchronic myocardial ischemia can also cause significant degradation incardiac function. If the ischemic episode is limited in severity orduration, then cardiomyocytes survive and are protected from furtherischemic insult through several preconditioning mechanisms. However,with acute and prolonged severe periods of ischemia, cardiomyocyte deathoccurs (Kloner R A, et al., Consequences of brief ischemia: stunning,preconditioning, and their clinical implications: part 1. Circ. 2001;104:2981-2989). Under normal conditions, adult human cardiomyocytes lackthe capability to regenerate, and over time, damaged myocardial cellsare replaced by connective scar tissue along with a compensatoryhypertrophy of the remaining viable cardiomyocytes (Gulbins H, et al.,Cell transplantation—A potential therapy for cardiac repair in thefuture? Heart Surg Forum 2002; Vol 5(4):E28-34). This replacement ofinfarct by scar tissue leads to a loss of functional myocardium withinthe ischemic area, a progressive remodeling of the non-ischemic area, orborder zone, and an overall reduction in cardiac performance.

Stroke is the third leading cause of death in the United States and thenumber one cause of adult disability. Ischemic stroke caused by bloodflow interruption to the brain due to blockage of an artery by a bloodclot accounts for about 70-80 percent of all strokes. A loss of bloodflow to the brain deprives an area of brain cells of oxygen andnutrients which results in cell death. Body functions controlled by thearea of the brain that has been damaged are lost. These functionsinclude speech, movement and memory.

Repair of injured tissue is a complex process that begins at the momentof injury and can continue for months to years. This process can bebroken down into three major phases; inflammatory, proliferative andremodeling. (Witte M B, et al. General principals of wound healing. SurgClin North Am 1997, 77:509-528).

The inflammatory phase is immediate and can last for 5 to 7 days. Duringthis phase, if there is tissue damage and/or cellular disruption as withtrauma, vasoconstriction occurs and a clot forms which serves as atemporary protective shield for the exposed or damaged tissues. The clotprovides cytokines and growth factors released by activated plateletsthat initiate the wound closure process and chemotactic signals torecruit circulating inflammatory cells to the wound site. Vasodilationfollows and phagocytosis is initiated.

The proliferative phase is next and can last up to three weeks. Duringthis phase granulation commences and involves the formation of a bed ofcollagen by fibroblasts which results in the filling of the defect. Newcapillaries are formed in a process called granulation tissue formation,which is followed by contraction in which the wound edges come togetherto reduce the lesion. The last stage of the proliferative phase isre-epithelialization. In skin wound healing, keratinocytes move in alldirections from a point of origin across a provisional matrix to coverthe wound.

The final phase of tissue repair is the remodeling phase. It can last upto two years and includes the production of new collagen which continuesto increase the tensile strength of the wound.

The immune system has been recognized as an important regulator oftissue repair. It is composed of two parts, humoral and cellulardefenses. The humoral arm includes antibodies and complement and littleis known about the role that this arm plays in the process of tissuerepair. On the other hand, much is known of the cellular arm whichincludes neutrophils, macrophages and T lymphocytes. These cellpopulations migrate into the wound in an ordered timeframe andcontribute to the repair process through the secretion of signalingmolecules in the forms of cytokines, lymphokines and growth factors.(Witte M B, et al., General principals of wound healing. Surg Clin NorthAm 1997, 77:509-528). Neutrophils are the first cells to appear at thewound site and are responsible for phagocytosis and debridement.Macrophages are the next cells to migrate into the wound. They completethe inflammatory and debridement processes and deliver critical tissuerepair cytokines and growth factors. T lymphocytes are the last cells tomigrate into the lesion and appear during the proliferative phase andtheir role includes the downregulation of the inflammatory response andgrowth state as this phase of the process concludes (Barbul A., Role ofT-cell-dependent immune system in wound healing. Prog Clin Biol Res1988, 266:161-175). Not all cells in the immune system are believed toparticipate in the tissue repair process. The role of B cells (Blymphocytes) in tissue repair, for example, is unclear and is presumedby those knowledgeable in the field to be inconsequential since helperT2 cell cytokines and B lymphocyte activating factors have not beendetected at the wound site. What little amount of evidence exists on therole of B cells in tissue repair suggests B cells have a pathogenic role(Zhang M, et al., Identification of a specific self-reactive IgMantibody that initiates ischemia/reperfusion injury. Proc Natl Acad SciUSA 2004, 101:3886-3891).

Instead, B cells are best known for the role they play in the productionof antibodies. They are generated from hematopoietic stem cells (HSCs)throughout life, first in the fetal liver and then in the adult bonemarrow. Cytoplasmic cascades are initiated in response to tissuemicroenvironment signals that result in altered expression of proteinsrequired for B cell maturation. The mature bone marrow B cell expressesIgD on its surface membrane which protects it from self antigen induceddeath. This mature cell moves into the periphery where it can beactivated by antigen to become either an antibody-secreting plasma cellor a memory B cell.

While treatment options available to patients who have lost tissuefunction have increased recently, these options remain limited in theireffectiveness. New therapies that can limit the amount of cell death andrestore loss of body function are greatly needed. An appealing conceptfor the treatment of tissue injury is cell-based therapy. Evidence ofcells engrafting into the damaged tissue coupled with an improvement offunction supports this approach. However, the most appropriate cell typehas yet to be defined. While many groups are eager to begin treatingpatients with various cells, researchers are just now beginning tounderstand some of the mechanisms of how these cells repair injuredtissue. What is needed is an identification of which cell, orcombination of cells, is most appropriate for the repair of damagedtissue.

While many different cell therapy methods are being tried, the commongoal in cell therapy is the introduction into injured tissue of a cellthat is either functionally related to the targeted tissue, such as withdelivery of skeletal myoblasts into damaged myocardium, or primordialcells (stem or progenitor cells) that are hoped will regenerate newtissue and structures thereby returning function to the injured organ.The bone marrow is a well understood source of stem cells for a varietyof tissue but primarily for the blood system. Early attention was givento the bone marrow as a source of potentially therapeutic cells afterseveral studies demonstrated that animals with labeled bone marrow cellsthat were subjected to a tissue injury such as a myocardial infarctionwere found to have some of these labeled bone marrow cells integratedinto the healing tissue. However, while integration of bone marrowderived cells into healed tissue was demonstrated, many questions remainunanswered including what cell type from the bone marrow integrated intothe tissue and the extent to which these cells contributed to thefunctional recovery of the injured tissue. Nonetheless, there wassufficient potential of a therapeutic effect for research to proceed inthis field including human experimentation. Experimentation into bonemarrow derived cell therapy has utilized either the entire bone marrow,also known as unfractionated bone marrow, or the isolation of theendothelial progenitor, hematopoietic (CD34+, AC133+) andnonhematopoietic (CDstro1+) stem cells contained within it. While theexperimental use of unfractionated bone marrow, bone marrow derivedprogenitor, and stem cells continues, early results from their use havebeen disappointing due to only modest improvement or negative outcomes,questioning the relevance of the earlier animal experimentation and thetherapeutic value of bone marrow derived cells.

SUMMARY OF THE INVENTION

The present invention addresses the problems described above byproviding compositions and methods for improving tissue functionfollowing injury. Our invention demonstrates for the first time thatcells of the B cell lineage including pre-pro-B cells, pro-B cells,pre-B cells, immature B cells, and some mature B cells, whentransplanted into damaged tissue have the ability to enhance recoveryfollowing injury.

It is to be understood that the methods of the present invention may beapplied to any animal. It is further to be understood that the term“animal” includes a “human” in the present application.

B cell therapy has the potential to promote repair in many differenttypes of tissues including but not limited to heart, brain, kidney,muscle and lung. It is a surprising finding of the present inventionthat administration of B lymphocytes to ischemic cardiac tissue improvesrecovery of cardiac function. B lymphocytes are preferred cells of thepresent invention for administration to the ischemic cardiac tissue.Injection of these cells improves cardiac function. The presentinvention reveals that administration of an effective amount of Blymphocytes or modified versions thereof improves the function ofinjured tissue. In a preferred embodiment, the present invention revealsthat administration of an effective amount of B lymphocytes or modifiedversions thereof improves cardiac function. Modified B cells include Bcells that are subjected to hypoxia or chemical changes, are geneticallymodified, or are otherwise altered by exposure to varying chemical orphysical conditions. These conditions may include but are not limited totemperature, pressure, osmotic conditions, pH, and varyingconcentrations of molecular compounds, electrolytes, and proteins suchas B cell activating factor, and similar agents.

A preferred type of cell which is administered to injured tissue is thebeta (B) lymphocyte and its precursors, hereinafter called a B cell or Blymphocyte. Any type of B cell may be used. B cells can be characterizedby the presence of specific surface proteins, as known to one ofordinary skill in the art. These include but are not limited to B220,CD19, CD43, CD45RA, CD5, Mac-1, IgM, IgD, IgG, CD62L, CD23, CD21, CD40and B cell receptor (Igαβ). In one embodiment the B cells are human Bcells and are characterized by having one or more CD19, B220 or B cellreceptor (Igαβ) surface proteins. The present invention describesstudies performed in rats using antibodies to the CD45RA antigen, whichis a member of the CD45 antigen family that also includes the CD45,CD45RB, and CD45RO antigens.

The present invention reveals that B cells used for treating injuredtissue should be relatively pure and should not contain appreciablenumbers of stem cells. In the present application, relatively pure meansat least 80% pure, 85% pure, 88% pure, or even higher degrees of puritysuch as at least 90% pure, preferably at least 95% pure, preferably atleast 97% pure, or preferably at least 98% pure as determined byfluorescence activated cell scan (FACS) analysis. The B cell populationin the bone marrow is heterogeneous, containing pre-pro-B cells, pro-Bcells, pre-B cells, immature B cells, and some mature B cells. In someexperiments reported in the present application, when the CD45RAantibody is used to isolate the B cells from the bone marrow of rats,all of these different types of B cells are obtained and used foradministration into the animal. While not wanting to be bound by thefollowing statement, it is believed that any type of B cell, orcombinations thereof, may be used in the method of the presentinvention.

B cells may be administered directly into the injured tissue, intotissue surrounding the injury, topically, intracerebroventricularly,intramuscularly, intramyocardially, intrarenally, intrahepatically orsystemically. In one embodiment, B cells may be administered intoinfarcted myocardium or into myocardium surrounding the infarctedtissue. Injection of cells into the ischemic cardiac tissue or throughthe cardiac vasculature enhances cardiac function following infarction.The methods of the present invention can be used for lessening thedecline in cardiac function following an ischemic episode. Further, themethods of the present invention improve cardiac function followingischemia or following ischemic injury. This improvement in cardiacfunction provides a better quality of life or longevity for suchindividuals, enabling them to engage in physical activities that mightbe otherwise proscribed or contraindicated. By restoring cardiacfunction following ischemia, use of the method of the present inventiondecreases the chance of cardiac arrest following an episode of cardiacischemia.

The present invention includes methods for increasing the concentrationof B cells at a desired site, such as injured tissue, by administering asubstance that binds to the B cell and also to a binding site in thevicinity of the injured tissue or to a binding site on a device locatedadjacent to the injured tissue. Such a substance may be a bifunctionalantibody that may, for example bind to CD19 antigens on B cells and alsoto a binding site located in the vicinity of the injured tissue or to abinding site on a device located adjacent to the injured tissue. In thismanner, the number of B cells in the vicinity of the injured tissue isincreased. This method may be used whether the B cells are resident inthe animal or the human, or are harvested, purified and administered tothe animal or the human.

The present method may be used to treat other conditions related to poorperfusion or less than normal blood flow and oxygenation, including butnot limited to the following: peripheral vascular disease, decreasedtissue perfusion in diabetics for example in tissue located in theextremities, decreased cardiac perfusion in patients withatherosclerosis of one or more coronary arteries, decreased cardiacperfusion in patients undergoing bypass surgery or another cardiacprocedure, renal disease including ischemic renal diseases, decreasedcranial perfusion in patients with atherosclerosis of one or morecarotid arteries or branches thereof, or with atherosclerosis of one ormore vertebral arteries or other arteries in the cerebrovascularcirculation, temporary ischemic episodes, stroke, occlusion of vesselsdue to trauma, a mass, or any other cause.

The present invention is not limited to treatment of ischemic tissue orcardiac tissue. B cells may be administered to any injured tissue.Trauma, disease, chemical or other environmental exposures are otherproximate causes. Ischemia is one type of condition that producesinjured tissue. Preferably, B cells are administered to tissue followingany injury. Tissue injury may result from many different causes. Forexample, tissue injury may occur following ischemia, hemorrhage, organtransplant, trauma, surgery, inflammation, infection, bums, diseaseprogression, aging or many other causes.

B cells may also be administered in conjunction with other forms oftherapy. Substances which may be co-administered include but are notlimited to the following: stem cell mobilizing agents (GM-CSF, SDF,GCSF, platelet-derived growth factor (PDGF)); growth factors VEGF, FGF,IGF-1; nitric oxide donors such as nitroglycerin; COX-2 inhibitors;diuretics, angiogenic factors (VEGF, angiostatin inhibitors); factorsthat enhance blood flow; anti-inflammatories; anti-hypertensives; HMGco-reductase inhibitors; statins; angiotensin converting enzyme (ACE)inhibitors; wound healing enhancers; NSAIDS; chemokine antagonists;thrombin; extracellular molecules; chemokines including but not limitedto CXCL12, CXCL13, CCL19, CCL21, CCL25, CXCL9, and CXCL10; integrinligands including but not limited to MADCAM1 (mucosal addressincell-adhesion molecule 1) and VCAM1 (vascular cell-adhesion molecule 1);interleukin-4; and factors including any environmental cues that enhancethe survival and effectiveness of the B lymphocytes or combinationsthereof.

It is believed that the present method may be used to enhance perfusionof less than adequately perfused tissue and to enhance a cell's abilityto withstand ischemic conditions, thereby preventing or substantiallydelaying the onset of damaging ischemic episodes. Such therapy is alsobeneficial in patients with sub-optimal perfusion of tissue such thatthe tissue performs better in situations of enhanced demand, forexample, during exercise.

While not wanting to be bound by the following statement, it is believedthat the administration of B cells to cardiac tissue may activateresident cardiac stem or progenitor cells in or near the infarctedcardiac tissue, enhance a cell's ability to withstand ischemicconditions, and inhibit the breakdown of collagen.

The present method may also be used for improving cardiac function inpatients who are not candidates for bypass surgery.

The present invention also provides kits which may be used to preparerelatively pure populations of B cells that may be used subsequently foradministration to ischemic tissue. Such kits include various antibodiesknown to one of ordinary skill in the art that are useful in selectingand separating desired cells from a heterogeneous population of cells.These antibodies generally comprise primary antibodies that recognizesurface antigens, such as proteins, polypeptides and glycoproteins thatare characteristic of specific cells and are known to one of ordinaryskill in the art. These kits may include such antibodies that recognizethese surface antigens. Directions for using a kit are enclosed witheach kit.

These kits may include materials and apparatus used in harvesting Bcells. For example, if the B cell is obtained from bone marrow, thesekits may include an 11 gauge tapered needle device designed to penetrateinto the interior of bone usually the posterior superior ilium butsometimes the sternum, iliac crest, tibia or femur. Once penetrated, apolycarbonate syringe is attached and a vacuum applied to obtain themarrow.

The kits include antibodies and materials that are specific for the celltype to be isolated, for example B cells, and may be further specializedfor use in the heterogeneous cell mixture obtained from a specifictissue or organ. Materials may include apparatus or reagents useful inseparation or preparation such as tubing conduits, separators,filtrators, and containers, incubation apparatus including tissueculture equipment and containers, and chemical or molecular reagents.That is, isolation of B cells from a cell preparation from bone marrowmay require the use of an antibody or antibodies specific for thesurface antigens on the bone marrow derived B cells. In addition, such akit may include an antibody or antibodies specific for the surfaceantigens on the bone marrow derived cells to be separated from thedesired bone marrow derived B cells. In this manner, a positiveselection technique may be used optionally in combination with anegative selection technique. In order to facilitate negative selection,kits may be designed to include antibodies that are specific for allcell types except for the type to be isolated, for example B cells, inorder to isolate the cell type through negative selection. The toolscontained in these kits may be constructed of materials such asplastics, stainless steel, nitinol, rubber and other materials thatbind, concentrate or exclude cells. The kits may also contain primaryantibodies that are labeled so that the labeled cells may be separatedby techniques know to one of ordinary skill, including but not limitedto immunomagnetic separation. Such labels are commercially available.These kits may optionally contain secondary antibodies, which themselvesmay be labeled, and which may bind to the primary antibodies. Labelsthat may be attached to antibodies are known to one of ordinary skill inthe art and include, but are not limited to, light emitting labels andmagnetically responsive labels. Antibodies may be stored in containersin the kits which may be customized for protection from light, heat orother undesirable conditions. Antibodies may be stored in a lyophilizedstate or in a convenient buffer system optionally containing apreservative. Kits may also contain pharmaceutically acceptable buffersfor use in handling samples during purification and elution steps, andfor suspension of the isolated B cells for storage or for subsequentadministration to the cell donor.

In one embodiment, kits may contain antibodies for positive selection,negative selection or both, in the form of affinity columns. Suchcolumns may contain antibodies bound at their Fc region to a matrixwithin the columns. Heterogeneous cell preparations may be introducedinto the affinity column.

These kits may also contain containers for mixing the antibodies withthe cell preparations, means to transfer solutions such as pipettingmeans, graduated flasks, graduated centrifuge tubes, and the like.Further, the kits may include prepackaged closed systems to insuresterility.

These kits may also contain devices for the delivery of the B cells. Avariety of devices may be included depending on the targeted deliverysite. Intravascular and transcutaneous delivery could be achieved withstandard syringes while delivery to the brain, heart and kidney mayinvolve specialized transluminal devices that allow for the infusion ordirect injection of cells into or around the targeted organ.

Accordingly, it is an object of the present invention to provide amethod to enhance recovery of function in injured tissue throughadministration of B cells to the injured tissue, in the vicinity of theinjured tissue or through other means leading to the injured tissue.

It is an object of the present invention to provide a method to enhancerecovery of cardiac function in injured cardiac tissue throughadministration of B cells to the injured cardiac tissue or in thevicinity of the injured cardiac tissue.

It is another object of the present invention to provide an effectiveamount of relatively pure B cells for administration to the injuredtissue or in the vicinity of the injured tissue.

Another object of the present invention is to provide kits useful forobtaining B cells and purifying B cells for subsequent administration toa human.

Another object of the present invention is to provide kits useful inseparation of B cells from other cells in a heterogeneous cellpopulation.

An advantage of the present invention is that injured tissue is improvedfollowing administration of B cells, resulting in enhanced function andquality of life, and decreased morbidity and mortality.

An advantage of the present invention is that cardiac function isimproved following administration of B cells to ischemic cardiac tissue,resulting in enhanced cardiovascular function and quality of life, anddecreased chance of cardiac arrest.

These and other objects, features and advantages of the presentinvention will become apparent after a review of the following detaileddescription of the disclosed embodiments and claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: Depicts flow cytometric separation of the ckit+ hematopoieticstem cells (HSC) and CD45RA positively selected cells (B cells).

FIG. 2: Primary screen monitoring cardiac contractility followinginjection of specific populations of bone marrow cells post ischemicinjury. Bone marrow was harvested from donor Sprague-Dawley rat femursand tibias and various cell populations isolated. Six recipient ratsunderwent ligation of the left anterior descending coronary artery andsaline, 1×10⁶ HSCs, 5×10⁵ HSCs combined with 5×10⁵ B cells, or 1×10⁶ Bcells alone were directly injected into the infarcted area. After 2weeks, the surviving animals (5 to 6 rats) underwent follow-upechocardiography and hemodynamic measurements including cardiaccontractility (dP/dT). Sham operated (no ligation) and ligation plussaline injection animals were included as controls. Note the degradationof the heart's contractility measured by +dP/dT as well as the heart'sability to relax measured by −dP/dT when HSCs combined with B cells areinjected and the improvement of these parameters when B cells alone areinjected.

FIG. 3: B cells reproducibly augment cardiac function following ischemicinjury. Bone marrow was harvested from donor Sprague-Dawley rat femursand tibias and B lineage and stem cell populations isolated. Therecipient rats underwent ligation of the left anterior descendingcoronary artery and a total of 1 million freshly isolated cells (HSC andB cells) or overnight cultured cells (Cultured B cells and B cellsco-cultured with non-hematopoietic stem cells (NHSCs)) were injectedimmediately after ligation into the infarcted area. After 2 weeks, thesurviving animals underwent follow-up echocardiography and hemodynamicmeasurements. Percent of ventricular short-axis diametric shortening wasmonitored as a measure of cardiac function and analysis of variance(ANOVA) was performed to determine data significance. Sham operated (noligation), ligation plus saline and HSC injection animals were includedas controls.

FIG. 4: Cardiac cell but not necessarily cardiomyocyte proliferation isup regulated following B cell injection post myocardial infarction.Animals were implanted with a BrdU pellet at time of infarction and cellinjection. After 2 weeks, animals were euthanized and hearts processedfor assessment of BrdU incorporation into the nuclear DNA ofproliferating cells. Positive cells were identified using antibodiesdirected against BrdU. The percentage of cells staining positive forBrdU was determined by comparison to the total number of cellsquantitated within the peri-infarct domain. Significance of the data wasassessed using the Student t-test, two-tailed.

FIG. 5: B cells preserve cardiac tissue following ischemic injury byreducing apoptosis. Assessment of cellular apoptosis was performed usingthe TUNEL assay. Animals underwent a ligation operation followed byeither saline or B cell injection (1 million cells). Animals wereeuthanized at 48 hours after surgery and hearts were processed forevaluation of apoptosis. Apoptotic cells within the peri-infarct domainwere quantitated by comparison to the total number of cells quantitatedin the peri-infarct domain. Significance of the data was assessed usingthe Student t-test, two-tailed.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a new, effective and efficient method fortreating injured tissue, through administration of an effective amountof B cells. Tissue injuries arise from numerous causes, including butnot limited to, ischemia, diabetes and other chronic diseases, organ andtissue transplant, trauma, burns, stroke (both ischemic andhemorrhagic), infection, inflammation, surgery and other causes. The Bcells to be administered are preferably autologous cells and areharvested from the donor using techniques known to one of ordinary skillin the art. Sources of such B cells are generally known and include, butare not limited to bone marrow, blood, spleen, lymph nodes, andallogeneic sources. B cells are then purified from the heterogeneouscell population in order to obtain a relatively pure population of Bcells. Such purification techniques are described in the presentapplication. Kits are provided to purify B cells from the heterogeneouscell populations.

The present invention provides for the use of B cells in the preparationof a medicament for administration to an animal or a human, wherein themedicament is useful in treating injured tissue. Such medicaments mayinclude a pharmaceutically acceptable carrier. Such medicaments mayoptionally include another substance that facilitates restoration offunction of the injured tissue or the function and/or survival of therelatively pure population of B cells administered to the animal or thehuman. It is to be understood that the methods of the present inventionmay be applied to any animal. It is to be understood that the term“animal” includes a “human” in the present application.

The B cells may be administered in conjunction with other therapiesknown to one of ordinary skill in the art for enhancing tissue repair orfor enhancing the retention, efficacy, and survival of the B cellsadministered to the damaged tissue. The present invention provides kitsuseful in the separation and purification of B cells from heterogeneouscell mixtures.

While the present invention may be used to treat different types ofdamaged tissue, cardiac tissue is a preferred tissue to be treated withthe present invention.

Preparation of B Cells

Any source of B cells may be used in the present invention. Such B cellsmay be derived from the bone marrow, spleen, lymph nodes, blood or otherallogeneic tissues that are sources of B cells, as known to one ofordinary skill in the art. Preferred sources of B cells are bone marrowand blood. References for such methods include: Funderud S, et al.,Functional properties of CD19+ B lymphocytes positively selected frombuffy coats by immunomagnetic separation. Eur J Immunol 199020(1):201-6; Monfalcone et al., Increase leukocyte diversity andresponsiveness to B-cell and T-cell mitogens in cell suspensionsprepared by enzymatically dissociating murine lymph nodes. J Leukoc Biol1986 39(6):617-28; and, Miltenyi Biotec sells kits with protocols toisolate B cells and plasma cells from tissue.

Using sterile techniques known to one of ordinary skill in the art, inone embodiment, bone marrow is preferably obtained from the posteriorsuperior ilium. The B cells obtained with the present method may beimmediately used after isolation and relative purification, may bestored for subsequent use, or may be cultured for a period of timebefore use. The B cell population in the bone marrow contains pre-pro-Bcells, pro-B cells, pre-B cells, immature B cells, and some mature Bcells. In the present application, the term B cell encompasses pre-pro-Bcells, pro-B cells, pre-B cells, immature B cells, and mature B cells.From blood or other tissues, B cells can be isolated using techniquesknown to one of ordinary skill in the art. The results of the presentinvention demonstrate that these B cells should be relatively free fromstem or progenitor cells when administered to a recipient.

Method to Obtain B Cells or Progenitor B Cells from Heterogeneous CellPopulations

Methods to obtain B cells or precursor B cells from heterogeneous cellpopulations are known to one of ordinary skill in the art. Many of thesetechniques employ primary antibodies that recognize molecules on thesurface of the desired B cells or B cell precursors and use theseantibodies to positively select these cells and separate them fromunwanted cells. This technique is known as positive selection. Othertechniques commonly employed by one of ordinary skill in the art useprimary antibodies that recognize molecules on the surface of the cellsto be separated from the desired B cells or B cell precursors. In thismanner, molecules on these unwanted cells are bound to these antiseraand these cells are removed from the heterogeneous cell population. Thistechnique is known as negative selection. A combination of positive andnegative selection techniques may be employed to obtain relatively purepopulations of B cells or precursor B cells. In the present application,relatively pure means at least 80% pure, 85% pure, 88% pure, or higherdegrees of purity such as at least 90% pure, at least 95% pure, at least97% pure, or at least 98% pure.

Numerous techniques are available to one of ordinary skill to separateantibodies bound to cells. Antibodies may be linked to various moleculesthat provide a label or tag that facilitates separation. In oneembodiment, primary antibodies may be linked to magnetic beads thatpermit separation in a magnetic field. In another embodiment, primaryantibodies may be linked to fluorescent molecules that permit separationin a fluorescent activated cell sorter. Fluorescent and magnetic labelsare commonly used on primary and/or secondary antibodies to achieveseparation. Secondary antibodies which bind to primary antibodies may belabeled with fluorescent molecules that permit separation of cells in afluorescence activated cell sorter. Alternatively, metallic microbeadsmay be linked to primary or secondary antibodies. In this manner,magnets may be used to isolate these antibodies and the cells bound tothem.

To achieve positive or negative selection, the heterogeneous cellpopulation is incubated with primary antibodies for a time sufficient toachieve binding of the antibodies to the antigen on the cell surface. Ifthe primary antibodies are labeled, separation may occur at this step.If secondary antibodies are employed, then the secondary (anti-primary)antibodies are incubated with the cells bound to the primary antibodiesfor a time sufficient to achieve binding of the secondary antibodies tothe primary antibodies. If the secondary antibody has a fluorescentlabel, then the cells are sent through a fluorescence activated cellsorter to isolate the labeled antisera bound to the desired cell. If thesecondary antibody has a magnetic label, then the selected cell with theprimary antibody and secondary antibody-labeled microbeads form acomplex that when passed through a magnet remain behind while the otherunlabeled cells are removed along with the cell medium. The positivelylabeled cells are then eluted and are ready for further processing.Negative selection is the collection of the unlabeled cells that havepassed through the magnetic field.

MACS Technology (Miltenyi Biotec) is an example of what may be used inthe present invention and is based on the use of MACS MicroBeads, MACSColumns and MACS Separators. This technology is known to one of ordinaryskill in the art. MACS MicroBeads are superparamagnetic particles thatare coupled to highly specific monoclonal antibodies. They are used tomagnetically label the target cell population. They are approximately 50nm in size, not visible with light microscopy, biodegradable, and gentleon cells.

As the MicroBeads are extremely small, the use of a high-gradientmagnetic field is required to retain the labeled cells. The MACS ColumnTechnology is specifically designed to generate this strong magneticfield while maintaining optimal cell viability and function. By using aMACS Column with a coated, cell-friendly matrix placed in a permanentmagnet, the MACS Separator, the magnetic force is now sufficient toretain the target cells labeled with a minimum of MicroBeads. By simplyrinsing the column with buffer, all the unlabeled cells are washed outthoroughly, without affecting the labeled or unlabeled cell fractions,thus ensuring optimal recovery. By removing the column from the magnet,the labeled fraction can be obtained. With MACS Technology both thelabeled and the unlabeled fraction are now highly pure, and an optimalrecovery of the cells is guaranteed.

Isolation of B cells from heterogeneous cell populations and stem cellpopulations involves the negative selection process in which the marrowfirst undergoes red cell lysis by placing the bone marrow in a hypotonicbuffer and centrifuging the red blood cells out of the buffer. The redblood cell debris remains in the supernatant which is removed from thetest-tube. The bone marrow derived cells are then resuspended in abuffer that has the appropriate conditions for binding antibody.Alternatively, the bone marrow can be subjected to a density gradientcentrifugation. The buffy coat layer containing the bone marrow derivedcells is removed from the gradient following the centrifugation. Thecells are washed and resuspended in the antibody binding buffer and isthen incubated with primary antibodies directed toward stem cells, Tcells, granulocytes and monocytes/macrophages (called lineage depletion)followed by positive selection using antibodies toward B cells.

In the present application anti-CD3 and anti-CD4 antibodies were usedfor T cells, anti-CD11b/c antibodies were used formonocytes/macrophages, anti-granulocyte antibodies were used forgranulocytes, and c-kit antibodies were used for stem cells. Then, forpositive selection, CD45RA antibodies were used for rat B cells.

In another embodiment, the primary antibodies are attached to a matrixand the cells are incubated with this matrix. Those cells with surfaceantigens recognized by the primary antibodies are bound to the primaryantibodies while other cells without these surface antigens are not. Inone embodiment, this matrix is contained in a syringe and acts as anaffinity column. Bound cells are subsequently eluted from the column andmay be used at this stage or subjected to a further purification step inanother affinity column containing the same primary antibody or anotherprimary antibody that recognizes another surface antigen on the targetcell. Elution of bound cells may occur using techniques such asadjustment of pH, addition of a buffer of altered tonicity, salt orother techniques useful for interfering with antigen-antibody bindingknown to one of ordinary skill in the art. These affinity columns may beused for positive selection, negative selection or both, in order toobtain a relatively pure preparation of B cells for administration tothe injured tissue.

Pretreatment of B Cells

B cells may be optionally pretreated by exposing them to hypoxicconditions in order to increase B cell adhesion to mesenchymal cells andto enhance B cell activity. This pretreatment can be achieved once thebone marrow is harvested and the B cells isolated as described earlier.Prior to delivery of the B cells, the B cells are incubated within aclosed system containing a sub-physiologic level of oxygen.

Number of Cells for Administration

The number of cells to be administered will be related to the area orvolume of injured tissue to be treated, the method of delivery, and insome cases the species to be treated. For example, rats have relativelysmall hearts and so the myocardium is much smaller and thinner thanlarger species such as dogs, pigs and humans. The number of cells usedin rat studies is described in Example 1 and in the Figures. Theinjections in the rat study reported herein, consisted of 4 injectionsof 20 ul each. Thus a total of 10⁶ cells in 80 ul was injected into rathearts weighing approximately 1.2 gm at termination.

One non-limiting range of B cell number for administration is 10⁴ to10¹⁴ B cells, depending on the volume of infarcted tissue to be treated,and in some cases the species to be treated. Other ranges include 10⁵ to10¹² B cells and 10⁶ to 10¹⁰ B cells.

Individual injection volumes can include a non-limiting range of from 1ul to 1000 ul, 1 ul to 500 ul, 10 ul to 250 ul, or 20 ul to 150 ul.Total injection volumes per animal range from 10 ul to 10 ml dependingon the species, the method of delivery and the volume of the tissue tobe treated.

For administration to larger animals higher number of cells in largervolumes may be employed. In a study performed using pig hearts, a totalof 10⁸ unfractionated bone marrow cells was administered in 16injections of 100 ul each in 1.6 ml total volume. Human patients will betreated with an effective amount of B cells which may be similar numbersof cells and similar volumes of cells as described above for use inpigs, but may be higher or lower.

Site and Method of Administration

B cells may be administered using any method that delivers the B cellsto the injured tissue or the tissue surrounding the injury. B cells maybe administered through injection via a syringe and appropriately sizedneedle.

B cells may also be administered through a cannula placed within a bodycavity, a vessel, a duct, a lumen of an organ, within an organ, a spacesurrounding an organ such as pericardial or pleural spaces, orintrathecally. B cells may also be applied topically for surface woundsor directly to injured tissue during surgery. In one embodiment, B cellsmay be administered through an intraarterial cannula to injured tissuesupplied by the artery.

In the present method the cells are administered directly into theinjured tissue in one or more injections. Cells may also be administeredinto the border zone surrounding injured tissue. In the case ofinfarcted cardiac tissue, B cells may also be administered directly intothe infarcted cardiac tissue and into the border zone surrounding theinfarcted cardiac tissue. In another embodiment, cells may beadministered into less than adequately perfused tissue which is notinfarcted. It is to be understood that the distance or spacing betweeninjections into the tissue will vary depending on the size of the areato receive cells and the species. For example, in rats the heart issmall and the injections of cells are spaced about 1 mm to 2 mm apart.

In pigs or humans with much larger hearts than rats, cells areoptionally administered into the myocardium using the Boston ScientificStiletto™ myocardial injection catheter with intracardiacechocardiography (ICE) guidance. The myocardial injection catheter isintroduced via femoral artery cutdown and used according to themanufacturer's instructions. In one embodiment, the total number ofcells is delivered to the tissue in 16 injections (8 injections to theischemic region and 8 injections into border zone region) of 100 μlvolume containing no less than 1×10⁸ cells in a total volume of about1.6 ml. Such treatment may comprise injections of freshly isolated bonemarrow cells into the injured area or border zone of the injured area.

Another method of the present invention is to give agents that cause Bcells to mobilize into the circulation and/or to home to the targetedtissue. These methods assist in achieving increased levels of B cells atthe targeted tissue without the need for harvest and reinjection. Forexample, CXCL13 is a known B cell chemokine that could be delivered tothe targeted tissue along with a B cell mobilizing agent to augmentpresence of B cells in the targeted tissue. Another technique utilizes achemokine antagonist that lowers the amount of chemokines containedwithin the targeted tissue to a level that is compatible with B cellactivity. Another method is to implant a substrate or device such as anintravascular stent into or near the targeted tissue wherein thesubstrate or device is coated with a matrix containing an antibody whichreacts with a B cell antigen thereby localizing and concentrating Bcells at the implant site. In this method, the number of circulating Bcells could be augmented through the harvest from another organ such asbone marrow, isolated, concentrated and delivered back to the patient'sblood system.

Another technique utilizes other methods to condition B cells in vivo topromote adherence of the B cell to the surface of the implantedsubstrate, device or injured tissue in order to concentrate or increasetheir levels at the targeted tissue. In this technique, a substancecould be delivered systemically, such as a bifunctional antibody, thatadheres to surface antigens on B cells, for example the CD19 surfaceantigen, and also adheres to the implant surface or injured tissue tocause B cell localization at that site. This approach can be used inconjunction with administration of autologous B cells harvested from theanimal or human. This approach can also be used in situations whereinautologous B cells are not harvested from the animal or human but whenan increase of endogenous B cells at the injured tissue is desired. Forexample, administration of such a substance to the animal or the humanwith the injured tissue can bind to available B cells, such ascirculating B cells, and also to the site of the injured tissue, therebyincreasing the number of B cells at the site.

Method of Administration to Infarcted Cardiac Tissue

Preferably, the injections are made through a small gauge needle,preferably in the range of 32 gauge to 21 gauge, or in a range of 30gauge to 23 gauge. The needle size may vary depending on the type,depth, and thickness of the tissue to be injected. For example in therat, a 27 gauge needle was employed. In humans, various gauges ofneedles or catheters may be used. The Boston Scientific Stiletto™myocardial injection catheter which utilizes a 27 gauge needle may beemployed. In one embodiment, the injections are made through thepericardium into the infarcted region of the myocardium.

All of the procedures associated with the harvest and injection of cellsare performed using sterile technique. Areas of infarction arevisualized using echocardiography and the injections are performed usingboth fluoroscopy and intracardiac echocardiographic guidance. Anothertechnique includes the use of trans-esophageal echo. Another techniqueincludes the use of visual, tactile and anatomical landmarks fortranscutaneous direct injection. Yet another technique reported isintravenous delivery which requires no visualization technique. Ifintra-coronary infusion is used, fluoroscopy is the preferred method.

In one method of cell delivery, an occlusive balloon is placed proximalto the infarcted tissue to deliver cells. A common device is a PTCAballoon that is inflated using low pressure and the cells are deliveredvia the wire lumen. The advantage is to temporarily stop flow to enhancecell adhesion and uptake within the targeted tissue.

In another embodiment, the injections are made through vascularcatheters equipped with injection means. Such catheters are guided byone of skill in the art, such as an interventional cardiologist, aveterinarian, or another trained assistant. The catheter is directed tothe infarcted region through the vascular system leading to the coronaryarteries using visualization techniques, including but not limited tothe intracardiac echocardiography guidance (ICE) and NOGA mapping (mapselectrical signal conductance) as known to one of ordinary skill in theart. NOGA is a commercial name for a catheter system that utilizes athree dimensional (3D) mapping system combined with an ECG detectionalgorithm that reportedly correlates the signal to myocardial viability.

Carriers

The B cells to be administered are suspended in a pharmaceuticallyacceptable carrier such as pharmaceutically acceptable fluid. Suchfluids include but are not limited to saline, cell culture medium andplasma. Additional pharmaceutically acceptable carriers includescaffolds, matrices, glues, gels and other tissue retention carrierswith or without cytokines and growth factors.

Timing of Administration of B Cells

The timing of administration of B cells to injured tissue will varydepending on the condition of the patient. In general, B cells areadministered in hours, days, weeks, months or years after an injury. Thepresent invention may also be used to treat chronic conditions in whichcase administration schedules will be determined by the physician orveterinarian. Cells may be administered to patients one or more timesfollowing injury depending on the severity of the patient's conditionand the recovery of function following administration of B cells.

Combination Therapy

In addition to the use of cells alone for the treatment of tissueinjury, the present invention contemplates the combination therapy ofcells with other substances. Such substances include, but are notlimited to, substances which facilitate restoration of tissue functionor the function and/or survival of the cells to be administered(delivery success). Such substances include but are not limited to thefollowing: growth factors VEGF, FGF, IGF-1; nitric oxide donors such asnitroglycerin; COX-2 inhibitors; anti-inflammatories; HMG co-reductaseinhibitors; statins; angiotensin converting enzyme (ACE) inhibitors,thrombin, chemokines including but not limited to CXCL12, CXCL13, CCL19,CCL21, CCL25, CXCL9, and CXCL10; NSAIDS, chemokine antagonists,thrombin, extracellular molecules, integrin ligands including but notlimited to MADCAM1 (mucosal addressin cell-adhesion molecule 1) andVCAM1 (vascular cell-adhesion molecule 1); interleukin-4; substancesaltering vascular perfusion such as vasodilators, osmotic agents,anticoagulants, acid/base modifying agents, surfactants, or combinationsthereof. These substances may be administered in dosages and usingregimens known to one of ordinary skill in the art.

Treatment of Injured Tissue

B cells may be administered to any injured or otherwise compromisedtissue. Preferably, B cells are administered to tissue following anyinjury. Tissue injury may result from many different causes. Diseases orconditions leading to injury of tissue that may be treated with themethod of the present invention include but are not limited to thefollowing: tissue injury following ischemia or hemorrhage, organtransplant, trauma, surgery, inflammation, infection, cardiovasculardisease, heart disease including acute infarction, chronic ischemia andmyocardium with less than desired perfusion; peripheral vasculardisease; decreased perfusion due to diabetes; kidney disease includingchronic renal ischemia; congestive heart failure; and, ischemic stroke.

B cells may be administered directly into the injured tissue and/or maybe administered into tissue surrounding the injury. In one embodiment, Bcells may be administered into infarcted myocardium or into myocardiumsurrounding the infarcted tissue. Injection of cells into the ischemiccardiac tissue enhances cardiac function following infarction. Themethods of the present invention can be used for lessening the declinein cardiac function following an ischemic episode. Further, the methodsof the present invention improve cardiac function following ischemia.This improvement in cardiac function provides a better quality of lifeor longevity for such individuals, enabling them to engage in physicalor other activities that might be otherwise proscribed orcontraindicated. By restoring cardiac function following ischemia, useof the method of the present invention decreases the chance of cardiacarrest following an episode of cardiac ischemia.

The present method may be used to treat other conditions related to poorperfusion or less than normal blood flow and oxygenation, including butnot limited to the following: peripheral vascular disease, decreasedtissue perfusion in diabetics for example in tissue located in theextremities, decreased cardiac perfusion in patients withatherosclerosis of one or more coronary arteries, decreased cardiacperfusion in patients undergoing bypass surgery or another cardiacprocedure, renal disease including ischemic renal diseases, decreasedcranial perfusion in patients with atherosclerosis of one or morecarotid arteries or branches thereof, or with atherosclerosis of one ormore vertebral arteries or other arteries in the cerebrovascularcirculation, temporary ischemic episodes, stroke, occlusion of vesselsdue to trauma, a mass or any other cause.

A System for Treating Injured Tissue in a Human

The present invention includes a system for treating injured tissue in ahuman. This system comprises several steps comprising: obtaining asample containing B cells from the human with the injured tissue;purifying the sample to produce a relatively pure population of B cells;combining the relatively pure population of B cells with apharmaceutically acceptable carrier to produce a composition; and,administering the composition to the human with the injured tissue in anamount effective to treat the injured tissue in the human.

In this system, equipment and reagents are employed to achieve the goalof treating the injured tissue in the human as described elsewhere inthis application.

Kits

The present invention includes kits for use in isolation of human Bcells. These kits are useful in the isolation of B cells fromheterogeneous cell populations. Instructions are provided for use of thekit.

Kits may employ positive selection techniques, negative selectiontechniques, or a combination thereof, to isolate a relatively purepopulation of B cells from a heterogeneous mixture of cells. Positiveselection techniques employ antibodies that recognize antigens on the Bcells. Antibodies that recognize antigens B cells include but are notlimited to antibodies that bind CD19, CD19+, B220+ or B cell receptor(Igαβ)+.

A non-limiting example of a kit for positive selection of B cells ispresented in Example 10. Negative selection techniques employ antibodiesthat recognize antigens on the cells to be separated from the B cells. Anon-limiting example of a kit for negative selection of B cells ispresented in Example 11. Negative selection techniques employ antibodiesthat include but are not limited to antibodies that bind the followinghuman cell surface antigens: CD2, CD3, CD14, CD16, CD36, CD43, CD56, andglycophorin A that reside on T cells, NK cells granulocytes,monocytes/macrophages, and erythrocytes.

Generally speaking, kits include a separation chamber, optionallyincluding: a centrifugation chamber; a collection bag connected to theseparation chamber; means to connect the collection bad to theseparation chamber such as connection units, connection lines, andluers; a manifold, antibodies that recognize antigens on B cells orantibodies that recognize antigens on non B cells, or both; separationmeans such as a filter or a column; and, a collection vial for theisolated B cells.

Antibodies may optionally be linked to magnetic beads as known to one ofordinary skill in the art. Kits may employ affinity columns in whichantibodies used for positive or negative selection are suspended in amedium such as a chromatography medium known to one of ordinary skill inthe art, for example Sepharose. Such antibodies have been describedelsewhere in this application. Such affinity columns may act asseparation chambers. Another separation chamber is a tube coated withantibodies on its inner wall.

Kits may also employ materials such as polystyrene, polypropylene,stainless steel, nitinol, rubber or other materials known to bind tocells.

Kits optionally contain buffers for elution of bound cells or cellstrapped by a filter, a buffer for resuspending the isolated B cellsbefore administration to the human. Filters that trap cells and separatecells from viruses or other undesired plasma components may also beemployed.

The following examples will serve to further illustrate the presentinvention without, at the same time, however, constituting anylimitation thereof. On the contrary, it is to be clearly understood thatresort may be had to various embodiments, modifications and equivalentsthereof which, after reading the description herein, may suggestthemselves to those skilled in the art without departing from the spiritof the invention.

EXAMPLE 1 Recovery of Cardiac Function Following Ischemia in Rats

Bone marrow cells were harvested from donor rats and separated into stemcell and B cell populations and directly injected into infarctedrecipient rat hearts. These animals are extremely in-bred and are oftenused in this way to substitute for the human autologous situation.Baseline and two-week follow-up echocardiography was performed tomeasure left ventricular end systolic and diastolic diameter and percentshortening (percent shortening is the heart's short-axis “strokediameter” (difference between end-diastolic and end-systolic diameters)divided by the diameter in diastole. This measurement accuratelyreflects global left ventricular function and is highly correlated withejection fraction.

A normal heart with normal wall motion and contractility will have agreater percent shortening value compared to a dysfunctional heart inwhich contractility is impaired and shortening is reduced). Hemodynamicindices including positive dP/dT was measured during the 2-weekfollow-up procedure. Hearts were harvested and subjected to histologicalstaining for determination of infarction area and capillary density.

The co-injection of stem cells and B cells had a deleterious effect onleft ventricular function compared to injection of B cells alone as seenwith the positive dP/dT measurement in FIG. 2. The deleterious effect ofstem cells combined with B cells is one possible explanation for thenegative results reported using whole bone marrow.

The results indicate that an improvement in cardiac function occurredwhen relatively pure populations of B cells were injected as determinedby percent shortening (see FIG. 3). This improvement was unique to Bcells since administration of HSCs alone did not enhance function nordid other cells contained within the bone marrow (data not shown). Ourstudies investigating B cell mechanism of action on cardiac ischemicinjury repair indicate that B cells both increase cellular proliferationand decrease cellular apoptosis. Taken together, these data demonstratethat administration of relatively pure populations of B cells provided afunctional and anatomical recovery in the heart.

Animals: Male Sprague-Dawley rats (about 250-350 g body weight) wereanesthetized with isoflurane (3.5% with 0.8 L oxygen), subjected toorotracheal intubation and ventilated with positive pressure (Topo, KentScientific). Anesthesia was maintained using isoflurane (3.5% with 0.5 Loxygen). Animals were then placed on a controlled heating pad maintainedat 37° C. (Gaymar T/Pump). ECG monitoring was performed in order toverify ST elevation associated with left ventricular infarction.

Bone marrow cell separation: Donor Sprague-Dawley rats were anesthetizedwith 80 mg/kg of ketamine, 2.0 mg/kg of xylazine then euthanized viaexsangination. Femurs and tibias were harvested and the shafts flushedwith Dulbecco's modified essential medium (DMEM) supplemented with 2%fetal bovine serum (FBS) and gentamicin (2 mg/ml). The bone marrow (BM)was collected and diluted 1:2 with Dulbecco's phosphate buffered saline(DPBS) and layered onto a Ficoll density gradient (Histopaque-1077,Sigma, St. Louis Mo.). The buffy coat was collected and washed 3 timeswith DPBS then incubated with various antibodies and subjected to theMiltenyi cell separation procedure. B cells were selected either bylineage depletion using antibodies including but not limited to CD3 (Tcells), CD8 (T cells), Granulocyte (neutrophils), CD11b/c (monocytes)and ckit (HSCs) antibodies followed by positive selection with CD45RAantibody or by positive selection with CD45RA antibody alone.Hematopoietic stem cells (HSCs) were selected by lineage depletion withthe antibodies mentioned above followed by positive selection with ckitantibody while non-hematopoietic stem cells were selected by lineagedepletion with the antibodies mentioned above plus CD45 antibodyfollowed by positive selection with antibody directed against ckit.Purity of cell populations were determined using flow cytometricanalysis. In the present application, relatively pure means at least 80%pure, 85% pure, 88% pure, or even higher degrees of purity such as atleast 90% pure, preferably at least 95% pure, preferably at least 97%pure, or preferably at least 98% pure. Once populations were separatedand purity confirmed by fluorescence activated cell scan analysis (FIG.1), cells were either kept at or 37° C. until the day of myocardialinjection or immediately injected into the animals. On the day of cellinjection, HSCs, NHSCs and B cells were washed with DPBS, andresuspended in sterile saline for injections.

Induction of myocardial infarction: After surgical preparation anddrape, using sterile technique and equipment, a left lateral thoracotomywas performed and a rib-spreading chest retractor was inserted. The leftanterior descending artery (LAD) was isolated and ligated using sterile6-0 prolene and the artery ligation was confirmed by the visual changeof color, or blanching in the area of infarction along with an ECGchange of ST elevation. Sham animals had the same procedure however theligature around the LAD was left untied. Another control group was thesham group plus administration of B cells. All groups are shown in thefigures.

The ST level was measured relative to the PR interval. With tachycardiaor ischemia the direction changes, as does the ST amplitude.Pathological processes that cause ST elevation shift the entire vectorfrom its long axis of the heart orientation and move it through the areaof transmural ischemia (infarction or spasm).

Bone marrow cell treatment: After ligation of the LAD and confirmationof myocardial infarction, animals received a total of 10⁶ cells (exceptfor the one case shown in FIG. 2 in which 5×10⁶ B cells alone wereinjected) in 4 injections of 20 ul (about 250,000 cells per injection,12,500 cells per ul) directly into the infarcted region are spaced about1 mm to 2 mm apart, using a Hamilton syringe with a 27 gauge needle.Injections were performed by the surgeon and done by hand, directly intothe myocardium. Control animals received saline. The chest was closedusing sterile suture (4-0 Vicryl) and the animal was allowed to recoverfrom the surgical procedure.

Cell Culturing Method

In another aspect of these experiments, animals received the B cellsthat have been cultured overnight at 37° C. In addition to culturingseparately, both stem cells and B cells were co-cultured with the Bcells placed on top of a transwell filter (0.4 um pore size) and thestem cells below the transwell filter. Cells were seeded at a 1:1 ratioof B cells to stem cells and cultured overnight in DMEM containing 10%FBS with 1% penicillin and 1% streptomycin. Either B cells or stem cellswere collected the next day and prepared for injections.

When B cells were co-administered with stem cells, the total number ofcells administered was 10⁶. Stem cells and B cells were mixed togetherand the slurry of cells was injected.

Echocardiographic evaluation: The echocardiographic procedure wasperformed immediately before opening the chest and repeated two weekslater at sacrifice. Initially, a 2-dimensional short-axis view of theleft ventricle (LV) was obtained at the level of the mid-papillary andapical levels. After optimizing gain settings and ensuring that theimage is on axis, M-mode tracings were recorded through the anterior andposterior LV walls. The M-mode echocardiogram provides a one-dimensionalview (depth) into the heart. The M-mode images represent echoes fromvarious tissue interfaces along the axis of the beam. These echoes,which move during the cardiac cycle, are swept across time on theoscilloscope/recording, providing the dimension of time. Thus, the lineswhich are seen on the recordings correspond to the position of theimaged structures in relation to the transducer and other cardiacstructures at any point in time. The M-mode echocardiograph uses a highsampling rate and can provide more accurate images of cardiac borders.Measurements of cardiac dimensions and motion throughout the cardiaccycle are often more accurately obtained from M-mode tracings. Thisorientation for M-mode tracings was chosen to allow delineation of wallthickness and motion in infarcted and noninfarcted regions. The resultswere recorded on VHS tapes, and the M-mode tracings analyzed. LV masswas been calculated automatically by the internal software using a cubeformula. Relative anterior wall thickness, relative posterior wallthickness, and LV internal dimensions were measured from at least threeconsecutive cardiac cycles. Endocardial fractional shortening and midwall fractional shortening was used as indices to estimate LV systolicfunction.

Restudy and Termination: After two weeks, animals were sedated andintubated as described above. A cutdown was performed to the cervicalarea and the right carotid artery was dissected. A 2 Fr Millar catheter(SPR-612 or SPC 320; Millar Instruments, Houston, Tex.) was insertedinto the LV through the right carotid. Pressure of the LV was recorded(dP/dT) along with aortic pressures. Animals were sacrificed with KCland the heart removed, wet weight recorded and processed forhistological evaluation.

Histological tissue processing: After sacrifice, the hearts were brieflyrinsed with saline perfusion and then fixed using 4% paraformaldehydevia a cannula through the aorta. Following fixation, the tissue wereprocessed one of two ways for analysis of cardiac cell apoptosis oranalysis of cardiac cell proliferation; 1) paraffin-embedded 2) embeddedin tissue freezing-medium.

Determination of cardiac cell proliferation: Animals were implanted witha bromodeoxyuridine (BrdU) pellet at the time of the myocardialinfarction and cell injection procedure. After 2 weeks, animals wereeuthanized and hearts processed for BrdU assessment. BrdU positive cellsare stained with labeled anti-BrdU antibodies and all nuclei arecounterstained with Hematoxylin. All images were taken using 40×objective lens and positive cells were quantitated. Few BrdU-positivecells were detected in sham (no ligation) operated animals. BrdUproliferating cells were observed in ligation plus saline treatedanimals, though more BrdU staining was found in the B cell treatedhearts (FIG. 4).

Determination of cardiac cell apoptosis: Animals underwent sham (noligation) operation or ligation with B cell injections. Animals wereeuthanized at 48 hours after surgery. Hearts were processed forhistological evaluation of apoptosis using the TUNEL assay. Apoptoticcells are stained with the stable chromogen, diaminobenzidine while allnuclei are stained with Hematoxylin. All images were taken using 40×objective and positive cells were quantitated (FIG. 5).

Statistics—Hemodynamic Data

T-tests were run and significance evaluated based on a two tailed testat p<0.05. Analysis of variance (ANOVA) was also performed on all thedifferent cardiac functional measurements and if differences in theLeast Squared Means between groups were detected, a Tukey post-hocanalysis was performed and the p-value given.

The results indicate that an improvement in cardiac function (seechanges in percent shortening FIG. 3) occurred when relatively purepopulations of B cells were injected. However, when stem cells and Bcells were co-administered, no improvement was observed. (FIGS. 2, 3).Freshly isolating the bone marrow cells immediately before injection andculturing produced the greatest improvement in cardiac functionfollowing injury (FIG. 3). Studies investigating B cell mechanism ofaction on cardiac ischemic injury repair indicate that B cells bothincrease cellular proliferation and decrease cellular apoptosis. Takentogether, these data demonstrate that administration of relatively purepopulations of B cells provided a functional and anatomical recovery inthe heart.

EXAMPLE 2 Treatment of a Human Patient Following Myocardial Infarction

A 55 year old male visits his cardiologist complaining of angina. Anarteriogram reveals 90% blockage in the left anterior descendingcoronary artery. During a treadmill test the patient suffers an acutemyocardial infarction and survives. Subsequent-tests reveal that aportion of the left ventricular myocardium is damaged from theinfarction.

The patient undergoes open-chest surgery to bypass the left anteriordescending coronary artery with a saphenous vein graft. During thesurgery, the infarcted area is visualized. Approximately 200 ml of bonemarrow are aspirated from the iliac crest and processed to separate outthe subpopulation of CD19+ cells. Sixteen injections of these bonemarrow derived B cells at a concentration of about 100 million cells per1.6 ml are made directly into the ischemic myocardial tissue in a totalvolume of about 100 ul per injection, using a 27 gauge needle.

Following completion of the surgery, the patient is monitored during thenext several months. The ischemic area which received injections ofCD19+ cells shows improvement in vascularity and contractile functionduring this period.

EXAMPLE 3 Treatment of a Human Patient Following Myocardial Infarction

A 62 year old female patient with an infarction of the right coronaryartery suffers ischemic damage. B cells are obtained from the patient'sbone marrow and cultured overnight before administration of the cells(1000 ul containing 120 million cells). The patient is placed on aregimen of drugs using a protocol similar to what is provided to a postinfarction patient by one of ordinary skill in the art. The next day,the patient's coronaries are cannulated with a guide catheter introducedat the groin via the femoral artery. A PTCA balloon is introducedthrough this catheter into the coronaries and inflated with sufficientpressure to occlude coronary blood flow. The PTCA wire is removed andthe cells are then injected through the balloon's wire lumen anddelivered distal to the inflated balloon. After approximately 90seconds, the balloon is deflated and the procedure is concluded.Following completion, the patient is monitored during the next severalmonths. The ischemic area which received injections of CD19+ cells showsimprovement in vascularity and contractile function during this period.

EXAMPLE 4 Treatment of a Human Patient Following Myocardial Infarction

A 35 year old obese male with an infarct in the left cardiac ventricleis indicated for a PTCA instead of a surgical bypass. The B cells areharvested from the iliac and delivered with a trans-femoral device thatcrosses the aortic valve, engages the left myocardium, deploys a needlethat allows for injection of the cells directly into the myocardium.Prior to injection, the B cells are mixed with a matrix substance suchas fibrin sealant, biopolymer or collagen that enhances cell retention,survivability and effectiveness. In addition, stents that are placed inthe proximity of the infarct are coated with a matrix and an antibodywhich reacts with a B cell antigen that preferentially attracts andretains these cells.

EXAMPLE 5 Treatment of a Human Patient with Congestive Heart FailureFollowing Ischemia

A patient suffers from congestive heart failure believed to be caused byischemia. The procedure begins with harvesting between 50 and 500 ml ofbone marrow from the patient's iliac crest and processed with a kit (aclosed-bag system) that isolates CD19+ cells in a relatively pureformulation. Prior to delivery of these cells, the patient's coronaryarteries are cannulated with a diagnostic catheter common to the PTCAindustry. Delivery of the purified cells into the artery is achievedwith injection of the cells into the diagnostic catheter. An improvementin cardiac function is measured days after administration of the cells.

EXAMPLE 6 Treatment of a Human Patient with Damage to the CentralNervous System Following Hemorrhagic Stroke

A patient suffers a hemorrhagic stroke due to bleeding from an arteriolebranching from the left middle cerebral artery. The bleeding in thevicinity of the postcentral gyrus threatens motor function. Theprocedure begins with harvesting between 50 and 500 ml of bone marrowfrom the patient's iliac crest and processed with a kit (a closed-bagsystem) that isolates approximately 200 million CD19+ B cells in arelatively pure formulation. Prior to delivery of these cells, thepatient's left internal carotid artery is cannulated with a diagnosticcatheter commonly known to interventional surgeons. A device is deployedinto the left internal carotid artery and threaded to the origin of themiddle cerebral artery. The purified B cells suspended in apharmaceutically acceptable carrier are slowly injected into the middlecerebral artery, and are carried by blood flow to the site ofhemorrhage. An improvement in motor function is measured days afteradministration of the B cells.

EXAMPLE 7 Treatment of a Human Patient with Damage to the CentralNervous System Following Hemorrhagic Stroke

A patient suffers a hemorrhagic stroke due to bleeding from an arteriolebranching from the posterior cerebral artery. The bleeding in thevicinity of areas 18 and 19 of the occipital cortex threatens visualfunction. The procedure begins with harvesting B cells from thepatient's blood and processing with a kit (a closed-bag system) thatisolates CD 19+ B cells in a relatively pure formulation. Prior todelivery of these cells, the patient's skull is stabilized in astereotaxic device and a craniotomy in the occipital bone is performedusing techniques commonly known to neurosurgeons. The dura is piercedand a needle connected to an injection syringe containing over 100million B cells in a pharmaceutically acceptable carrier is introducedinto area 18 surrounding the clot using a micromanipulator. B cells arethen slowly injected in a volume of 1-5 ul. This procedure is repeatedfor injection into area 19 surrounding the clot. An improvement invisual function is measured days after administration of the B cells.

EXAMPLE 8 Treatment of a Human Patient with Damage to the KidneysFollowing a PTCA Procedure

A patient undergoes a routine procedure to revascularize the myocardiumusing PTCA techniques including the use of contrast medium forfluoroscopic visualization. The patient's kidneys react to the contrastmedium causing portions of the kidney to fail referred to as contrastinduced nephropathy. The procedure begins with harvesting between 50 and500 ml of bone marrow from the patient's posterior ilium and processedwith a kit (a closed-bag system) that isolates CD19+ cells in arelatively pure formulation. Prior to delivery of these cells, thepatient's renal arteries are cannulated with a delivery catheterdesigned specifically for delivery of solutions to the renal arteries.Delivery of the purified cells into the artery is achieved withinjection of the cells into the catheter. An improvement in renalfunction is observed over the next several weeks after administration ofthe B cells.

EXAMPLE 9 Treatment of a Human Patient Undergoing Kidney Transplantation

A patient suffering from end stage renal disease undergoestransplantation of a kidney from a tissue matched donor. Prior toimplantation, between 50 and 500 ml of bone marrow is harvested from thepatient's posterior ilium and processed with a kit (a closed-bag system)that isolates CD19+ cells in a relatively pure formulation. Theseisolated B cells are then injected throughout the donor kidney prior tosurgically implanting the kidney. An improvement in renal function andorgan rejection is observed over the next several weeks afteradministration of the B cells.

EXAMPLE 10 Isolation of B Cells from Human Bone Marrow

In this example, bone marrow is chosen as the source of B cells and isselected by positive immunomagnetic separation. Human autologous bonemarrow B cells are isolated in a specially designed separation chamberwith collection bags attached that enable both fluid transfer and theseparation process in a closed and sterile environment. All materialsused in this B cell selection process are sterile. The collected bonemarrow is loaded into the separation chamber where it is diluted 1:2with phosphate buffered saline (PBS) and then layered onto a Ficolldensity gradient contained within the centrifugation section of thechamber. The bone marrow cells are subjected to density gradientcentrifugation. The upper Ficoll layer is directed towards a discardbag. The bone marrow mononuclear cells (MNCs) contained within theintermediate layer are detected by optical sensors, collected and washedwith PBS. The washed MNCs are directed towards a collection bagcontaining antibody directed against the B cell specific antigen, CD 19,and conjugated to a micromagnetic bead. The cells are incubated with theantibody-micromagnetic bead at a controlled temperature for 20 minutes.A magnet is then applied to the collection bag so that the cellsattached to the antibody-micromagnetic bead are held against the side ofthe collection bag. The cells that did not bind to the antibody areremoved. The magnet is then moved away from the bag and the cells boundto the antibody-micromagnetic beads are suspended in buffer containing apeptide recognized by the CD19 that displaces the cells from theantibody. A magnet is again applied to the bag and the released CD19+ Bcells are removed and collected. The B cells are washed onto a sterilefilter that separates the cells from unbound peptide which flows throughthe filter into a discard tube. The B cells are resuspended in bufferand are ready for transplantation back into the patient.

Materials (All materials are Sterile)

Some of the materials used in this example include a centrifugationchamber, stopcock manifold, connection units, connection lines, port forinput product, ports for washing solutions, ports for resuspension, portfor output product with needle injection site, needleless luers,collection bags or tube, waste bags, input line breakaway, vials forfinal product, 0.2 um filters, phosphate buffered saline (PBS), celltransplantation buffer, Ficoll solution, and CD19 antibody conjugated tomicromagnetic beads.

EXAMPLE 11 Isolation of B Cells from Human Blood

In this example, whole blood is chosen as the source of B cells and isselected by negative affinity separation. Human autologous peripheralblood B cells are isolated in a specially designed separation chamberwith collection bags attached that enable both fluid transfer and theseparation process in a closed and sterile environment. All materialsused in this B cell selection process are sterile. The collected bloodis loaded into the separation chamber where it is directed towards thecentrifugation chamber. Following centrifugation of the blood cells, theupper layer containing plasma is directed towards a discard bag. Theenriched leukocytes contained within the intermediate layer, the buffycoat, are detected by optical sensors and are directed towards acollection bag. The cells are washed with PBS and resuspended in buffercontaining antibodies directed against non-B cell specific antigensconjugated to CNBr-activated Sepharose. The antibodies used are directedagainst the following human cell surface antigens: CD2, CD3, CD14, CD16,CD36, CD43, CD56, and glycophorin A that reside on T cells, NK cellsgranulocytes, monocytes/macrophages, and erythrocytes. The cell-antibodymixture is incubated at a controlled temperature for 20 minutes and thenloaded into an empty column. Elution buffer is added to the column andthe cells not bound to antibody, the B cells, are eluted. The B cellsare washed with PBS, resuspended in buffer and are ready fortransplantation back into the patient.

Materials (All materials are Sterile)

Some of the materials used in this example include a centrifugationchamber, plastic column with stopcock, stopcock manifold, connectionunits, connection lines, port for input product, ports for washingsolutions, ports for resuspension, port for output product with needleinjection site, needleless luers, collection bags, waste bags, inputline breakaway, vials for final product, phosphate buffered saline(PBS), cell elution buffer, cell transplantation buffer, cell lineagespecific antibodies (anti-CD2, CD3, CD14, CD16, CD36, CD43, CD56, andglycophorin A) conjugated to CNBr-activated Sepharose.

All patents, publications and abstracts cited above are incorporatedherein by reference in their entirety. It should be understood that theforegoing relates only to preferred embodiments of the present inventionand that numerous modifications or alterations may be made thereinwithout departing from the spirit and the scope of the present inventionas defined in the following claims.

1. A method of treating injured tissue in an animal or a humancomprising administering an amount of a composition comprising arelatively pure population of B cells in a pharmaceutically acceptablecarrier to the animal or the human with the injured tissue, wherein theamount is effective to treat the injured tissue.
 2. The method of claim1, wherein the relatively pure population of the B cells is prepared bya method comprising substantially removing stem cells from aheterogeneous population of cells containing the stem cells and the Bcells to create a relatively pure population of the B cells.
 3. A methodof treating injured tissue in an animal or a human comprisingadministering a substance to the animal or the human that increases Bcell concentration at the injured tissue in the animal or the human. 4.The method of claim 1, further comprising administering a substancewhich facilitates restoration of function of the injured tissue,function of the relatively pure population of B cells or survival of therelatively pure population of B cells administered to the animal or thehuman.
 5. The method of claim 3, further comprising administering asubstance which facilitates restoration of function of the injuredtissue, function of the B cells or survival of the B cells administeredto the animal or the human.
 6. The B cells of claim 1, wherein the Bcells are derived from bone marrow, lymph nodes, spleen or blood.
 7. TheB cells of claim 3, wherein the B cells are derived from bone marrow,lymph nodes, spleen or blood.
 8. The B cells of claim 1, wherein the Bcells are CD19+, B220+ or B cell receptor (Igαβ)+ cells, or acombination thereof.
 9. The B cells of claim 3, wherein the B cells areCD19+, B220+ or B cell receptor (Igαβ)+ cells, or a combination thereof.10. The injured tissue of claim 1, wherein the injured tissue isischemic tissue.
 11. The injured tissue of claim 10, wherein theischemic tissue is ischemic cardiac tissue.
 12. The injured tissue ofclaim 3, wherein the injured tissue is ischemic tissue.
 13. The injuredtissue of claim 12, wherein the ischemic tissue is ischemic cardiactissue.
 14. The injured tissue of claim 1, wherein the injured tissue iscardiac, pulmonary, neural, hepatic, muscular or renal tissue.
 15. Theinjured tissue of claim 3, wherein the injured tissue is cardiac,pulmonary, neural, hepatic, muscular or renal tissue.
 16. A system fortreating injured tissue in an animal or a human comprising: obtaining asample containing B cells from the animal or the human with the injuredtissue; purifying the sample to produce a relatively pure population ofB cells; combining the relatively pure population of B cells with apharmaceutically acceptable carrier to produce a composition; and,administering the composition to the animal or the human with theinjured tissue in an amount effective to treat the injured tissue in theanimal or the human.
 17. A kit useful for isolating human B cells from aheterogeneous population of human cells comprising: a separation chambercontaining a centrifugation chamber; connection units; connection lines;a collection bag; antibodies capable of recognizing antigens on thehuman B cells, wherein the antibodies are linked to magnetic particles;a filter; a collection tube; and, instructions for using the kit.
 18. Akit useful for isolating human B cells from a heterogeneous populationof human cells comprising: a separation chamber containing acentrifugation chamber; connection units; connection lines; a collectionbag; antibodies capable of recognizing antigens on the cells in theheterogeneous population of human cells that are not B cells, whereinthe antibodies are linked to a medium; a collection tube; and,instructions for using the kit.
 19. The kit of claim 17 furthercomprising buffer for resuspending isolated B cells.
 20. The kit ofclaim 18 further comprising buffer for resuspending isolated B cells.